1. Field of the Invention
This invention relates generally to selection of hematopoietic cells. Specifically, this invention relates to selection of hematopoietic progenitor and hematopoietic stem cells using a mutant dihydrofolate reductase in combination with a nucleoside transport inhibitor and an antifolate.
2. Background Art
Retroviral-mediated gene transfer is a potential therapeutic strategy for a number of diseases that affect the hematopoietic system. (Karisson S. xe2x80x9cTreatment of genetic defects in hematopoietic cell function by gene transfer.xe2x80x9d Blood 78:2481 (1991)). The early hematopoietic cells including hematopoietic progenitor cells and hematopoietic stem cells (HSC) are desirable targets for gene therapy. The hematopoietic stem cell is especially desirable for gene therapy because it can contribute progeny to all hematopoietic lineages and can support hematopoiesis throughout the lifetime of an animal. Despite these attractive features, primate HSCs remain relatively refractory to genetic modification. Although retroviral vectors provide one of the best methods for HSC transduction, recent clinical trials have shown that current protocols result in very low levels of HSC gene transfer. (Dunbar et al. xe2x80x9cRetrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraft ment after autologous transplantation.xe2x80x9d Blood 85:3048 (1995) and Brenner et al. xe2x80x9cGene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.xe2x80x9d Lancet 342:1134 (1993)) Because the overall proportion of modified cells ranges from 10 to 0.01% after hematopoietic reconstitution, there are insufficient numbers of modified cells to be therapeutically effective. Thus, gene transfer is currently not a feasible treatment option for many diseases such as hemoglobinopathies, AIDS, chronic granulomatous disease, and cancer.
Because of the problem of low numbers of modified cells after hematopoietic reconstitution, selection of primitive HSCs is required for significant enrichment of modified cells. Selection of more differentiated cells would allow only a transient enrichment of modified cells due to the limited self-renewal capacity of these more mature cells.
One means of selecting modified cells that has been the focus of intense investigation involves dihydrofolate reductase. Dihydrofolate reductase is a ubiquitous cellular enzyme that catalyzes the generation of tetrahydrofolate, a necessary cofactor for purine and pyrimidine biosynthesis. Antifolate drugs such as methotrexate (MTX) are powerful inhibitors of DNA synthesis by virtue of their strong binding to the active site of DHFR. The discovery that single amino acid substitutions in the active site of DHFR could disrupt drug binding and thereby confer antifolate resistance (Simonsen et al. xe2x80x9cIsolation and expression of an altered mouse dihydrofolate reductase cDNA.xe2x80x9d Proc. Anti. Acad. Sci. U S A 80:2495 (1983)) raised the possibility that mutant DHFRs could potentially be used as drug resistance genes.
Mutant DHFR genes were the first drug resistance genes to be transferred to primary hematopoietic cells. Despite the theoretical advantages for using DHFR as an in vivo selectable marker, and the potential protection of hematopoiesis conferred by DHPR variants, evidence for in vivo selection has been equivocal using this experimental system. In MTX-treated mice containing the murine L22R (leucine to arginine substitution at codon 22) variant of DHFR, there appeared to be an enrichment of vector-transduced CFU-S cells following MTX treatment. (Corey et al. xe2x80x9cSerial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells.xe2x80x9d Blood 75:337 (1990)) However, when Southern blot analysis was done to confirm an enrichment of vector-modified cells in myeloid tissue, no such enrichment could be documented. The authors suggested that more prolonged MTX exposure may be required for in vivo selection of DHFR-modified immature hematopoietic cells, thus implying that cell cycle status may play a significant role in antifolate resistance. In analogous experiments done using a human DHFR variant, only a 2-fold increase in vector-expressing myeloid progenitor cells was seen following MTX treatment. (Zhao et al. xe2x80x9cLong-term protection of recipient mice from lethal doses of methotrexate by marrow infected with a double-copy vector retrovirus containing a mutant dihydrofolate reductase.xe2x80x9d Cancer Gene Therapy 1:27 (1994)) Hence, the authors conclude from this study that a modest level of in vivo selection of DHFR-modified progenitors can be accomplished utilizing MTX alone. However, several important controls were omitted from these experiments, such as the percentage of MTX resistant progenitors from DHFR mice that were not treated with MTX, so that even this modest level of selection remains unconvincing.
Another study reports that human myeloid progenitor cells transduced with a vector expressing a human DHFR variant were selected and expanded in vitro in MTX-containing cultures. (Flasshove et al. xe2x80x9cEx vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer.xe2x80x9d Blood 85:566 (1995)) Vector-expressing progenitor cells were amplified two-fold using this approach. However, this approach was in vitro. Further, it is not clear if transduced hematopoietic stem cells could be amplified using this method.
Consistent with these disappointing results, recent findings demonstrate that hematopoietic stem cells and progenitors are highly resistant to antifolates. (Blau et al. xe2x80x9cCytokine prestimulation as a strategy for in vivo selection: resistance of hemopoietic progenitors to folate analogs.xe2x80x9d Stem Cell Gene Therapy: Biology and Techniques. Sep. 28-Oct. 1 (1995)) These findings lead the authors to conclude that antifolates are poorly suited for the in vivo selection of transduced hematopoietic progenitor and stem cells. (Blau et al.).
The present invention overcomes these problems by disclosing a method which allows for effective elimination of unmodified hematopoietic cells which do not contain a transferred DHFR. The method thereby allows the modified hematopoietic progenitor and stem cells containing a modified DHFR to form a large proportion of the hematopoietic cells after reconstitution. To accomplish this result, the present method utilizes a nucleoside transport inhibitor to sensitize the non-modified hematopoietic cells to the antifolate. This invention, therefore, solves the problems identified by Blau et al. using a completely different approach.
xe2x80x9cThe use of nucleoside transport inhibitors has previously been proposed to potentiate the sensitivity of tumors to a variety of antifolates, including PALA, methotrexate, 5-fluorouracil, and acivicin, and potentially make these more effective by blocking the salvage of exogenous nucleosides. However, this use has produced perplexing results. It has been well demonstrated in cultured cell lines that exogenously added nucleosides can reverse the toxicity of these drugs and that nucleoside transport inhibitors such as NBMPR and dipyridamole can restore toxicity. (Marina et al. xe2x80x9cEffect of nucleoside transport inhibitors on thymidine salvage and the toxicity of nucleoside analogs in mouse bone marrow granulocyte-macrophage progenitor cells.xe2x80x9d Cancer Communications 3:367 (1991)) However, in vivo studies with mice have shown either no antitumor effect of the transport inhibitor, no potentiation of the antitumor effect, or an increased somatic toxicity with little change in the therapeutic index. In addition, clinical phase I and II studies combining dipyridamole with acivicin, methotrexate, 5-fluorouracil, or PALA have revealed only limited responses.xe2x80x9d
Thus, this invention utilizes for the first time nucleoside transport inhibitors to select against unmodified hematopoietic progenitor and stem cells to provide an effective means to utilize gene therapy to treat many diseases of hematopoietic cells.
In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, provides a method of in vivo selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells in a subject comprising genetically modifying hematopoietic progenitor cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which, when expressed, can confer antifolate resistance, administering to the subject the genetically modified hematopoietic progenitor cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the genetically modified hematopoietic progenitor cells.
The invention further provides a method of in vivo selection for hematopoietic progenitor cells genetically modified to contain and express a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic cells in a subject comprising administering to the subject the genetically modified hematopoietic progenitor cells, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the genetically modified hematopoietic progenitor cells.
In another aspect, the invention provides a method of in vitro selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells comprising genetically modifying hematopoietic progenitor cells by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to hematopoietic cells comprising the genetically modified hematopoietic progenitor cells and nonmodified hematopoietic cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic cells a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vitro for the genetically modified hematopoietic progenitor cells.
In another aspect, the invention provides a method of in vitro selecting for genetically modified hematopoietic progenitor cells containing and expressing a nucleic acid comprising a sequence encoding an antifolate resistant dihydrofolate reductase from nonmodified hematopoietic cells comprising administering to hematopoietic cells comprising genetically modified hematopoietic progenitor cells and nonmodified hematopoietic cells an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset by nucleoside salvage, and administering to the hematopoietic cells a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vitro for the genetically modified hematopoietic progenitor cells.
In yet another aspect, the invention provides a method of in vivo selection for genetically modified hematopoietic progenitor cells from nonmodified hematopoietic cells in a subject comprising, genetically modifying hematopoietic progenitor cells in the subject by introducing into the cells a nucleic acid comprising a sequence encoding a mutant dihydrofolate reductase which when expressed can confer antifolate resistance, administering to the subject an antifolate in an amount which inhibits the growth of the nonmodified hematopoietic cells, wherein the inhibition of the nonmodified hematopoietic cells by the antifolate can be offset in vivo by nucleoside salvage, and administering to the subject a suitable nucleoside transport inhibitor in an amount effective to prevent the offset of the inhibitory effect of the antifolate in the nonmodified hematopoietic cells, whereby the combination of the antifolate and the nucleoside transport inhibitor selects in vivo for the genetically modified hematopoietic progenitor cells.
These selection methods are applicable to not only hematopoietic progenitor cells, but to hematopoietic stem cells as well. Additionally, the nucleic acid comprising a sequence encoding a dihydrofolate reductase which is relatively resistant to an antifolate can further comprise a heterologous gene. Using a nucleic acid further comprising a heterologous gene, the methods of the present invention can be used in gene therapy procedures.
The present invention further provides nucleic acids encoding mutant dihydrofolate reductases and cells expressing those nucleic acids.